Background and purpose — Little is known about the long-term migration pattern of cementless stems in total hip arthroplasty (THA). Furthermore, the role of bioactive coatings in fixation, and thus migration, remains uncertain. Hydroxyapatite (HA) is the most commonly used bioactive coating. However, delamination of the coating might induce loosening. Alternatively, fluorapatite (FA) has proved to be more thermostable than HA, thereby potentially increasing longevity. We assessed the long-term migration of cementless stems with different coatings using radiostereometric analysis (RSA), thereby establishing a reference for acceptable migration.Patients and methods — 61 THAs in 53 patients were randomized to receive either a HA, FA, or uncoated Mallory-Head Porous stem during the years 1992 to 1994. Primary outcome was stem migration measured using RSA and secondary outcome was the Harris Hip Score (HHS). Evaluation took place preoperatively and postoperatively on the second day, at 6, 12, 25 and 52 weeks, and annually thereafter. At the 25-year follow-up, 12 patients (17 THAs) had died and 1 patient (1 THA) was lost to follow-up. Due to the high number of missing second-day postoperative RSA radiographs, the 1-year postoperative RSA radiograph was used as baseline for the comparative analyses.Results — Mean follow-up was 17 years (SD 6.6). All stems showed initial rapid migration with median subsidence of 0.2 mm (–0.1 to 0.6) and median retroversion of 0.9° (–3.2 to 2) at 12 months, followed by stable migration reaching a plateau phase. No stem was revised, albeit 1 stem showed continuous subsidence up to 1.5 mm. Comparing the different coatings, we could not find a statistically significant difference in overall 25-year migration . Median subsidence at 15-year follow-up was for HA –0.1 mm (–0.4 to 0.2), for FA 0 mm (–0.1 to 0.2), and for uncoated stems 0.2 mm (–0.1 to 0.5). Median internal rotation at 15-year follow-up was for HA not available, for FA 1.1° (–0.5 to 2.6), and for uncoated stems 0° (–0.5 to 0.4). HHS were also comparable , with at 15-year follow-up for HA 85 points (41–99), for FA 76 points (61–90), and for uncoated stems 79 points (74–90).Interpretation — The long-term migration pattern of cementless stems using different bioactive coatings has not previously been described. No beneficial effect, or side effect at long-term follow-up of bioactive coatings, was found. The provided migration data can be used in future research to establish thresholds for acceptable migration patterns cementless stem designs. Long-term migration data on cementless femoral stems in total hip arthroplasty (THA) is scarce, with only a few studies reporting migration measured with radiostereometric analysis (RSA) with follow-up beyond 10 years  possible at only 1 institute . Subsequently, it was decided to continue as a single-center study performed at the Leiden University Medical Center. From May 1992 to May 1994, all consecutive patients scheduled to receive a cementless primary THA for osteoarthritis, either primary or secondary to a systematic inflammatory disease and younger than 65 years of age, were approached for participation in a randomized, clinical RSA study.Included patients were randomized to 2 intervention groups receiving either an HA- or FA-coated implant and the control group received an implant without a bioactive coating. Treatment allocation was randomized with the use of a computer-generated randomization scheme and bilateral cases were allowed. The study design was single-blind; surgeons were aware of the coating used; clinical observers were blinded to the type of coating. The study was performed in compliance with the Helsinki Declaration, approval of the institutional medical ethical board was obtained, and all patients gave written informed consent.All THAs were implanted by experienced hip surgeons, or under their direct supervision. Surgeries were performed through a direct lateral approach in the lateral decubital position, except for 2 posterolateral approaches. For RSA measurements, 1-mm tantalum markers were inserted into the proximal femur during surgery. All patients received the same rehabilitation program starting with passive and controlled active movements on the first postoperative day and mobilization with full weight-bearing on the second postoperative day, after the first RSA radiograph was obtained.All patients received a Mallory-Head Porous stem with a dual tapered design with a round cross-sectional geometry . The stem is characterized by an anterior and posterior flange and wide lateral fin. It is made of a titanium alloy (Ti-6A1-4V), with a porous coating on the proximal third, a grit-blasted surface on the middle third, and a smooth satin-textured surface on the distal third . The impPatients were evaluated preoperatively and postoperatively at 6 weeks, 3 months, 6 months, 1 year, and annually thereafter. At each evaluation, RSA radiographs were obtained and the Harris Hip Score (HSS) was determined. Conventional anteroposterior and lateral radiographs were acquired preoperatively, at 6 weeks, and at 2, 5, 10, 20, 25 years postoperatively, and on indication . On the 6-week postoperative radiographs the stem orientation  was determined. Patients unable to attend follow-up moments were contacted to check implant status and whether implant-related problems had arisen.RSA radiographs were obtained using a uniplanar setup with the patient in supine position and the calibration cage underneath the examination table. During follow-up, in 2002, the initial calibration box  was replaced by a new box . Furthermore, in 2004 digital radiography was introduced. Both changes had no effect on the accuracy of the RSA measurements ; measured values of non-normally distributed data are reported as the median (range). Estimates are reported as the mean and the 95% confidence interval (CI). Reported analyses were performed according to the per-protocol principle to reflect the genuine effect of treatment . To safeguard for attrition bias, all analyses were repeated according to the intention-to-treat principle and compared with the outcomes of the per-protocol analyses. Migration and increase in HHS throughout the follow-up period were analyzed with use of a linear mixed model (LMM) with subject as a random effect. This model deals effectively with repeated measurements, missing values, and variation in duration of follow-up  (grant agreement number 248693) and the Dutch Arthritis Association  were received in support of this study. None of these sponsors took part in the design or performance of the study, neither in the collection, management, analysis, nor in the interpretation of the data; or in preparation, review, or approval of the manuscript. None of the authors has any potential financial conflict of interest related to this manuscript.From May 1992 to May 1994, 75 consecutive THAs in 67 patients were assessed for inclusion and 61 THAs in 53 patients were randomized . In 19 TSecond-day postoperative RSA radiographs were available in only 10 out of 42 stems, whereas RSA radiographs at 1-year follow-up were available in 38 out of 42 stems . As the Supplementary data). Furthermore, we could not find a significant difference in time to stabilization and subsequent migration, that is, no evidence of interaction . Migration of the stems was comparable among the 3 coatings at the prespecified time points of 5 years and 15 years postoperatively . Between-group differences did not change significantly over time . Adjusted analyses for age, sex, diagnosis, BMI, and postoperative HHS gave similar results. Furthermore, comparing the HHS at the 15-year follow-up point did not yield any statistically significant differences  with a liner revision 13 years after follow-up, the cup was revised due to aseptic loosening 20 years after follow-up and subsequently re-revised a couple weeks later due to malpositioning. In addition, 2 more cups in 2 THAs (coating unknown) were revised due to aseptic loosening. During follow-up, 12 patients (17 THAs) died due to causes unrelated to the THA. One patient (1 THA) was lost to follow-up due to emigration, the fate of this THA could not be determined.We found stable migration and thus fixation of the cementless Mallory-Head Porous stem over a period of 25 years. After initial migration, all but 1 of 42 stems stabilized after 1-year follow-up and there were no stem revisions. 4 cups were revised due to aseptic loosening and 13 liners were revised due to wear. Comparative analyses did not yield a difference in migration and clinical scores among HA, FA, and uncoated cementless stems. Furthermore, there was no difference in time to stabilization between coated and uncoated stems, thus excluding a delamination problem occurring later in follow-up.To our knowledge, this is the first long-term RSA study with over 20 years’ follow-up, and the first RSA study comparing migration of HA, FA, and uncoated stems. Only a few RSA studies describe migration beyond 10-year follow-up. Sesselman et al. (2018) reported migration of 26 cementless Cerafit stems with a follow-up of 10 years. They found a median subsidence of 0.01 mm at 2-year and 0.09 mm at 10-year follow-up, with most of the subsidence occurring during the first 6 postoperative weeks. Critchley et al.  reportedSøballe et al.  and KärrSeveral reviews have been published comparing HA-coated implants with uncoated implants. Gandhi et al.  reportedA recent registry study found an overall lower risk of stem revision for any reason for HA-coated stems compared with a non-HA coated stem; however, the rate for stem revision for HA-coated Mallory-Head stems was higher compared with the non-HA-coated counterpart (0.11% vs. 0.02%)  showed median subsidence of 0.2 mm (range 0.4–4.8) at 5 years’ follow-up of uncoated stems. In the current study we found the same median subsidence at 5 years’ follow-up but the range in this study was considerably smaller, with the largest subsidence being only 0.4 mm. Insufficient data on initial migration during the first postoperative year in this study might explain this difference.The cementless Mallory-Head Porous stem has an excellent 10-year survival record with 48 stem revisions of 5,932 primary THAs in the Dutch Arthroplasty Register LROI  and 27 sAll stems in our study showed stable subsidence, except for 1. This stem, with an untraceable coating, was inserted because of severe osteoarthritis at the age of 61 years. There was no preoperative template available but the postoperative radiograph showed a varus position of the stem with insufficient contact with the lateral cortex at the metaphysis, suggesting undersizing. Albeit that initial subsidence was unknown, the stem showed progressive subsidence from the 1-year follow-up onwards. On the 17-year follow-up radiograph there was obvious subsidence visible, next to radiolucencies and pedestal formation. Remarkably, this patient never scored less than 90 points on the HHS scale and at the 17-year follow-up moment the patient was asymptomatic, although, aged 78 years, she walked only about 200 meters.Overall, there was rather stable migration up to 14 years reaching a plateau phase, but thereafter migration patterns began to show greater variability, which especially holds for subsidence. From then onwards, patient attendance for regular follow-up also decreased dramatically, resulting in only 5 stems being available for analysis at 20 years’ follow-up. This low number of available, analog RSA radiographs in combination with relative low precision compared with the modern RSA technique are the most probable reasons for the great variability in stem migration patterns  took care of study logistics and RSA radiograph analyses without secretarial assistance. Hence, patients missing a follow-up moment were noticed only weeks later. These technical and logistical shortcomings resulted in the exclusion of 19 THAs. Furthermore, stem allocation to the randomization groups was not adequately documented and implant stickers of the manufacturer were missing or lacking essential information. The latter might have been related to the distinctive manufacturing process for stems in the current study; the attachment of 3 RSA markers and applying 3 different coatings might have interfered with regular application of implant stickers.This study has several limitations. First, there were too few migration measurements available during the first postoperative year to make meaningful analyses using the second-day postoperative radiograph as baseline, which is the conventional manner to calculate migration over time. However, as stems susceptible to failure will typically show progressive migration, using the 1-year postoperative RSA radiograph will also detect stems prone to failure. Second, the given coating could not be verified in 18 stems due to due to missing or insufficient implant stickers. To overcome this problem of unknown coatings, both per-protocol and intention-to-treat analyses were performed. The latter reflects the base case scenario, assuming all stems received the coating as per randomization. This is a plausible assumption as only 1 of 24 known coatings received a different coating as per randomization. Third, the Mallory-Head Porous stem is nowadays seldom used, limiting the clinical applicability of this study.In conclusion, this study could not establish a beneficial effect at long-term follow-up of bioactive coatings on migration, and thus fixation, in this type of stem. Neither could delamination of the less thermostable HA coating be proven. This study provides value migration data that can be used to establish an acceptable migration pattern of cementless stems with which new stem designs can be compared.Click here for additional data file.


Autologous hematopoietic cell transplantation (auto-HCT) may be performed in multiple myeloma (MM) patients relapsing after a previous auto-HCT. For those without an adequate dose of stored stem cells, remobilization is necessary. This retrospective study included patients who, following disease relapse after the first auto-HCT(s), underwent stem cell remobilization and auto-HCT performed using these cells. There were 305 patients, 68% male, median age at salvage auto-HCT was 59 years. The median time to relapse after the first-line penultimate auto-HCT(s) was 30.6 months, the median follow-up after salvage auto-HCT 31 months. The 2- and 4-year non-relapse mortality (NRM) after the salvage auto-HCT was 5 and 9%, the relapse incidence 56 and 76%, respectively. Overall survival (OS) after 2 and 4 years was 76 and 52%, progression-free survival (PFS) 39 and 15%. In multivariable analysis an increasing interval between the penultimate auto-HCT and relapse was associated with better OS and PFS, later calendar year of salvage auto-HCT with better OS. In conclusion, salvage auto-HCT performed with cells remobilized after a previous auto-HCT was associated with acceptable NRM. The leading cause of failure was disease progression of MM, which correlated with a shorter interval from the penultimate auto-HCT to the first relapse. Autologous hematopoietic cell transplantation (auto-HCT) is the standard of care in the treatment of eligible patients with multiple myeloma. It is usually incorporated into the first-line treatment, either as a single or a tandem auto-HCT , 2. DespFor patients considered suitable for a salvage auto-HCT, there may, however, be either an insufficient remaining stem cell dose or none at all in storage. Remobilization to procure new cells is then required. However, data on the efficacy of remobilization are scarce –8. AlthoResearch dedicated to this group is extremely scarce , 4, as aThe study was performed on behalf of the Chronic Malignancies Working Party (CMWP) of the EBMT. EBMT is a voluntary organization comprising more than 500 transplant centres from Europe and beyond. Accreditation as a member center requires submission of minimal essential data (MED-A form) from all consecutive patients to a central database.Member centers were invited to provide additional study-specific data about eligible patients, specifically information about stem cell collection, conditioning, and remobilization of stem cells. EBMT Centres commit to obtain informed consent according to the local regulations applicable at the time in order to report pseudonimysed data to the EBMT.This study was a retrospective analysis of all myeloma patients who had had an auto-HCT (single or tandem) and who, following disease progression, went on to undergo stem cell remobilization and an auto-HCT performed with cells procured during the remobilization (“new cells”). Patients who received a mixture of new cells and cells mobilized before the first auto-HCT and stored afterward were also eligible. Only patients whose interval between the penultimate auto-HCT and the subsequent remobilization was longer than six months were considered eligible. The analysis includes salvage auto-HCTs performed between 2000 and 2018.The first auto-HCT was defined as the initial auto-HCT performed for patients first-line, while the second auto-HCT as the last of the two tandem auto-HCTs performed in frontline. The penultimate auto-HCT was the last auto-HCT performed prior to the salvage auto-HCT, meaning that it could be either the first auto-HCT or the second auto-HCT, depending on the clinical situation.The primary objective of the study was to assess non-relapse mortality (NRM). The secondary objectives were to examine timing of recovery, overall survival (OS), relapse incidence (RI), progression free survival (PFS), the cumulative incidence of t-MDS and t-AML (t-MDS/t-AML CI), and the cumulative incidence of other secondary primary malignancies (SPM CI).All time-to-event outcomes were computed from the day of the salvage auto-HCT given after remobilization. OS was defined as the time from salvage auto-HCT to death from any cause and PFS was defined as the time from auto-HCT to relapse or progressive disease or death from any cause, whichever came first. Time to t-MDS/t-AML was defined as the time from salvage auto-HCT to therapy-related myelodysplastic syndrome or acute myeloid leukemia. Time to SPM was similarly defined, whilst time to any secondary malignancy was defined as the time to the first occurrence of either t-MDS/t-AML or SPM after salvage auto-HCT.The Kaplan–Meier estimator and log-rank test were used for OS and PFS, and the crude cumulative incidence estimator and Gray’s test were used for competing events . The median follow-up was calculated using the reverse Kaplan–Meier estimator .n = 130). There was only one extra event discovered in those patients compared to the data already available in the EBMT database. Based on this information, the data quality was found reliable enough to extrapolate and report the results for the whole cohort of patients.The quality of data for calculating the cumulative incidence of t-MDS/t-AML and other SPM was checked for patients for whom additional data was obtained  model with a random effect for each patient. This allowed inclusion of patients contributing data to only one of the two transplantations. Timing of recovery was compared between the first, second (if a tandem auto transplantation was performed), and salvage transplantation using Cox proportional hazard frailty models (including a gamma-distributed random effect for each patient) and using the exact method for tied observation times. We also compared timing of recovery after the salvage auto-HCT between patients who were infused with a mixture of old and new CD34 cells and those who were infused with only new CD34 cells using Kaplan–Meier plots and the log-rank test.P-values < 0.05 were considered significant. All estimates are reported with accompanying 95% confidence intervals in brackets. All analyses were performed in R version 3.6.3 [on 3.6.3 , using ‘Three hundred and five patients, fulfilling the inclusion criteria, were included in the analysis. Additional data requests allowed for more detailed characterization of a subgroup of 130 patients transplanted in 28 centres, including data on remobilization, collection yield, and the type of cells infused, as described in the section on Data Source in Materials and Methods. Patients with additional data obtained on remobilization and salvage auto-HCT were more likely to be diagnosed and transplanted more recently in comparison to patients for whom no additional data was obtained Table .Table 1P6 CD34 + cells/ kg body weight compared to 4.81 (range 1.63–38.4) x 106 CD34 + cells/ kg body weight obtained before the first auto-HCT (p < 0.0001). Further data on remobilization is presented in Supplementary Table There were 207 (68%) males and the median age at salvage auto-HCT was 59 (range 32–78) years. Median age at salvage auto-HCT increased with later calendar years Table . Patient6 CD34+ cells/ kg body weight. Data on the type of stem cells were available for 111 patients, among whom 96 (86.5%) received solely new cells, and 15 (13.5%) both old and new cells. Engraftment failure was noted in seven (2%) patients. All these patients eventually survived beyond 7 months. Five of them experienced a relapse. In the analyzed group, there were also 2 patients who died before engraftment within the first 28 days, and 2 for whom the follow-up was censored at 10 and 11 days before they reached engraftment. These patients were not included in the calculation of the engratment failure ratio.Salvage auto-HCT was performed after single agent high-dose melphalan conditioning in 292 (96%) patients, and melphalan in combination with other drugs in a further 12 (4%) patients. The source of hematopoietic stem cells was the peripheral blood in 293 (96%) patients, and patients received a median dose of 2.92 (1.07–24.5) x 10p = 0.43), while there was some evidence for a small difference between the second and salvage auto-HCT . Similarly there was no difference in time to platelet recovery > 20 × 109/ L between the first and the salvage transplantation , while there was again a difference between the second and the salvage auto-HCT . A significantly longer time to platelet recovery ≥ 50 × 109/L after the salvage auto-HCT in comparison to the first and second auto-HCT should be interpreted with caution due to frequent missing data resulting probably from the ambulatory follow-up at this stage of patients’ treatment. Data on hematopoietic recovery is presented in Table Time to neutrophil recovery did not differ between the first auto-HCT and salvage auto-HCT , while time to platelet recovery was unaffected by the type of cells infused .Patients who received a mixture of old and new cells had a shorter time to neutrophil recovery . Earlier platelet recovery to > 20 × 109/L was associated with higher number of infused CD34+ cells . Similarly, platelet recovery to > 50 × 109/L was dependent on the number of infused CD34+ cells .The number of infused CD34+ cells did not affect neutrophil recovery , the 2-, 4- and 6-year non-relapse mortality rates were 5% , 9%  and 10% , respectively. The 100-day and 1-year NRM were 1%  and 2% , respectively. The cumulative relapse incidence rates at 2, 4 and 6 years were 56% , 76%  and 81% , respectively; see Fig. The median OS was 51 months . OS probabilities were 76% , 52%  and 34%  at 2, 4 and 6 years, respectively. The median PFS was 17 months . PFS rates at 2, 4 and 6 years were 39% , 15%  and 9% , respectively; see Fig. p = 0.002 and 0.90 , p = 0.009 and year of salvage auto-HCT for OS with hazard ratio equal to 0.95 , p = 0.005.In univariable analysis, the time interval between the penultimate auto-HCT and relapse of greater than 30 months, being the median interval between the penultimate auto-HCT and relapse, was associated with survival benefit in terms of both OS and PFS and a slightly lower relapse incidence. Similarly, the calendar year of salvage auto-HCT impacted both OS and PFS, with patients transplanted before 2005 having inferior outcome. Male gender was associated with lower NRM, but a higher relapse incidence, resulting in no difference in survival. Patients with Durie-Salmon stage III disease at diagnosis had shorter OS. Other factors analyzed were not significantly associated with outcomes in univariable analysis. The detailed results of univariable prognostic factors analysis are presented in Table Infectious complications occurred in 126 patients (50%) among 250 for whom the data was available, with eight patients (6.3%) in this group succumbing to infection. Among 138 reported non-infectious complications, mucositis was the most frequent .Twenty out of 303 patients with data on secondary malignancy status available developed secondary malignancies after salvage auto-HCT: five acute leukemias, five myelodysplastic syndromes, one myelodysplastic/ myeloproliferative neoplasm  and seven solid tumors at a median of 34 months. One patient was diagnosed with a lymphoma, and for the remaining patient, the type of a secondary malignancy was not reported. This translated into a cumulative incidence of t-MDS/t-AML of 1% , 3%  and 4%  at 2, 4 and 6 years, respectively. The cumulative incidence of other SPMs was 1% , 3%  and 3% , respectively; see Fig. p = 0.055) as Supplementary Table There was a trend for patients with Durie Salmon stage III disease at diagnosis to have a higher incidence of t-MDS/t-AML (6% (95% CI 2–11) vs. 0% (95% CI 0–0) at 4 years, 7% (95% CI 2–13) vs. 1% (95% CI 0–4) at 6 years, This retrospective study analyzed the outcomes seen in auto-HCT performed using cells remobilized after a previous hematopoietic cell transplantation. This is the biggest study to date reporting data on this specific patients’ population.9/L did not differ between the first and the salvage auto-HCT. Surprisingly, there was a difference between the second and the salvage auto-HCT, with shorter time to hematopoietic recovery after the second auto-HCT. The reason for this finding is unclear and perhaps results from small patient numbers or from selection bias in those assigned to tandem auto-HCT. As reported previously, [9/L. This finding should, however, be interpreted with caution due to frequent missing data. Delayed platelet engraftment also was observed by Jimenez-Zepeda [Importantly, all but 2% of the patients treated with this approach engrafted. Time to neutrophil and platelet recovery ≥20 x 10viously, , 4 we obz-Zepeda , who repThe non-relapse mortality, which was 2%, 5%, 9%, and 10% at 1, 2, 4, and 6 years, respectively, and solely 1% after 100 days, seems acceptable. It is however not possible to compare the NRM obtained in this report with that reported by others, as many studies used both old and new stem cells but reported the outcomes for both types of cells together e.g., ; alterna2) was not associated with a higher incidence of secondary primary malignancies [The cumulative incidence of t-MDS/t-AML was 1%, 3%, and 4% after 2, 4, and 6 years, respectively , while for other non-myeloid secondary primary malignancies it was 1%, 3% and 3% respectively. This data indicates that the risk of secondary myeloid malignancies is not excessively high, although not negligible, especially taking into consideration the possibility of underreporting by the centres and intermediate follow-up of our study. Although the cumulative incidence of t-MDS/t-AML was higher than the rate of 1.4% at 6 years reported in the prospective observational CALM (Collaboration to Collect Autologous Transplant outcome in Lymphoma and Myeloma) study by the EBMT , it was gnancies . It mustgnancies . Therefognancies .Interestingly, although the difference did not reach statistical significance, it is worth noting that all secondary malignancies developed in patients who received only new cells. Whether this is a true observation and results from the accumulation of genetic mutations within stem cells in the course of multiple myeloma treatment or is, alternatively, a chance observation merits further study. The calendar year of salvage auto-HCT was significantly associated with the development of secondary malignancies, with patients transplanted before 2005 having the lowest cumulative incidence of both t-MDS/t-AML or any secondary malignancy. This may result from the fact that in this group of patients, the incidence of death before the development of secondary malignancies was almost twice as high as in the later years, reflecting poorer general outcomes following auto-HCT during these earlier years. The increase in median age at auto-HCT over time also may contribute to the increase in the incidence of secondary malignancies.It is difficult to speculate, how the efficacy of salvage auto-HCT performed with stem cells remobilized after the previous auto-HCT compares with the efficacy of salvage auto-HCT performed with stem cells harvested before the first-line auto-HCT, especially taking into consideration, that for a significant number of studies the information on the type of stem cells used is lacking , 27–32. Limitations of the study are its retrospective nature, the possibility of underreporting of the cumulative incidence of t-MDS/t-AML and other SPM, the lack of information on the previous treatment received including novel agents, the lack of cytogenetic data which is likely to impact outcome after salvage-HCT. Because only patients who proceeded to a salvage auto-HCT were selected we do not know how many patients there were with failed remobilization attempts. Additionally, the information on the number of CD34+ cells collected during remobilization is difficult to interpret, because it cannot be excluded, that in some patients with remaining stored stem cells from the first harvest, the collection was stopped earlier at the salvage remobilization. Nevertheless, we believe the study provides important and new information on the safety of salvage auto-HCT performed with cells procured after previous high-dose therapy.In conclusion, salvage auto-HCT performed with stem cells procured during remobilization after a previous auto-HCT is a viable treatment option when not enough stem cells are cryopreserved. The efficacy is comparable to the efficacy of salvage auto-HCT performed with stem cells harvested before the first auto-HCT and stored afterwards. The non-relapse mortality is acceptable, as are the incidence of secondary malignancies, both t-MDS/t-AML and solid tumors. The leading cause of failure was progression of multiple myeloma, which was associated with shorter time from penultimate auto-HCT to the first relapse.Table S1, Table S2, Table S3


E. coli, S. aureus, and P. aeruginosa).The need for biodegradable and biocompatible polymers is growing quickly, particularly in the biomedical and environmental industries. Cellulose acetate, a natural polysaccharide, can be taken from plants and modified with polycaprolactone to improve its characteristics for a number of uses, including biomedical applications and food packaging. Cellulose acetate-g-polycaprolactone was prepared by a three-step reaction: First, polymerization of ε-caprolactone via ring-opening polymerization (ROP) reaction using 2-hydroxyethyl methacrylate (HEMA) and functionalization of polycaprolactone(PCL) by introducing NCO on the hydroxyl end of the HEMA-PCL using hexamethyl lenediisocyanate(HDI) were carried out. Then, the NCO–HEMA-PCL was grafted onto cellulose acetate (using the “grafting to” method). The polycaprolactone grafted cellulose acetate was confirmed by FTIR, the thermal characteristics of the copolymers were investigated by DSC and TGA, and the hydrophobicity was analyzed via water CA measurement. Introducing NCO-PCL to cellulose acetate increased the thermal stability. The contact angle of the unreacted PCL was higher than that of cellulose acetate-g-PCL, and it increased when the chain length increased. The CA-g-PCL50, CA-g-PCL100, and CA-g-PCL200 showed very high inhibition zones for all three bacteria tested ( The demand for degradable and biocompatible polymers is rapidly increasing, especially in the biomedical and environmental sectors. These materials are available in large quantities, and they possess numerous advantageous properties . HoweverPolysaccharides are a group of carbohydrates with a large polymeric oligosaccharide formed through glycosidic bonds in the presence of multiple monosaccharides . In natul-lactide (PLLA), poly ε-caprolactone (PCL), polylactic–glycolic acid (PLGA) copolymers, and polyhydroxy–alkanoates (PHA) are classified as biodegradable synthetic polymers. Within the class of synthetic materials, PCL has recently drawn much attention for biomedical applications, including bone tissue engineering , of PCL samples in a DMF solvent. In Huggins’ method, intrinsic viscosity [η] is defined as the ratio of the increase in relative viscosity (rds zero .(3)ηredThe Kraemer method uses a similar approach, except the Kraemer equation is defined in terms of the natural logarithm of the relative viscosity :(4)ηinh=The measurement results are presented in Molar mass and viscosity could be related in an empirical manner through the Mark–Houwink–Sakurada (M–H–S) equation: (5)[η]=kThe intrinsic viscosity [η] according to Naar et al. equation is: (6)[η]=, k and a parameters are defined in the literature  at T = 3Mathematically, we find that:Therefore:Hydroxyl values of HEMA-PCLs were measured according to AOAC Official Method . In ordeFrom the definition of the hydroxyl value, which is the amount of KOH (in milligrams) needed to neutralize the acetic acid formed from the acetylation of 1 g of sample, the following relation is deduced: (12)Mn=5From the results obtained, it can be seen that this method yields acceptable results for polymers of relatively low molar mass.The results of the hydroxyl value measurement for the three samples are shown in From the tables, it can be seen that the results obtained by the viscosity measurement of molar mass and by the titration of hydroxyl value  are in agreement with the theoretical results and those found in the literature , i.e., tThe three HEMA-PCL-NCO samples were prepared by bulk reaction between hydroxyl-terminated HEMA-PCL and HDI, and the reaction is shown in By introducing NCO groups onto the HEMA-PCL terminals, new sharp peaks at 2270 re shown .The FTIR spectrum of HEMA-PCL-NCO prepared with HDI showed a slightly increased peak, around 780 from HDI .The CA-g-PCL copolymer was prepared by reacting HEMA-PCL-NCO with cellulose acetate. The reaction of copolymerization is shown in The FTIR spectra of CA-g-PCL copolymers are shown in The percent grafting and product yield of CA-g-PCL copolymers are shown in The yield was calculated using the relation:The analysis of results in The yield was relatively constantly over 88%, indicating that the HEMA-PCL-NCO added to the mixture was effectively incorporated into the CA.The thermal transition behavior of CA-g-PCL copolymers measured by TGA and DSC is shown in As shown in As shown in The contact angles (CAs) of the surface of CA-g-PCL were measured to determine the effect of PCL DP on the hydrophobicity of the following samples: PCL, CA-g-PCL50, CA-g-PCL100, and CA-g-PCL200.The results showed that the angles were 86.9°, 58.2°, 59°, and 62.5°, respectively .The cellulose acetate decreased the surface contact angle because it is hydrophilic, but by increasing the chain length of PCL, the contact angle increased because the PCL is hydrophobic.In order to study the morphology of CA-g-PCL, scanning electron microscopy (SEM) analysis was performed. The SEM image showed that the surface of cellulose Acetate (CA) changed, as illustrated in E. coli, P. aeruginosa, and S. aureus, using the solid medium disk diffusion method. This involved soaking paper discs with a volume of 20 µL of the colloidal solution of the three copolymers.The antibacterial activity of CA-g-PCL50, CA-g-PCL100, and CA-g-PCL200 were tested against three pathogenic bacteria, Two negative and positive antibiotic discs were used as controls. A disc was impregnated with DMSO solvent used for the dispersion of powders. The prepared wells were incubated with moderate agitation at 37 °C for 24 h. The test was carried out twice at the rate of three wells/sample each time.E. coli, S. aureus, and P. aeruginosa were chosen because they are the most common bacteria found in bone infections, such as osteomyelitis. The antibacterial activity of the prepared samples against the three bacteria is shown in E. coli, S. aureus, and P. aeruginosa). This antibacterial activity may be due to the presence of biomolecules in the samples [CA-g-PCL50, CA-g-PCL100, and CA-g-PCL200 showed a large high inhibition zone for all three bacteria , tetrahydrofuran (THF), hexamethylene diisocyanate (HDI 98%), methanol (MeOH), dichloromethane (DCM), dimethyl sulfoxide (DMSO), toluene, dibutyltin dilaurate (DBTDL), and dimethylformamide (DMF) were purchased from Sigma-Aldrich .2 (2 wt% of monomer) was added to the reaction flask. The reaction was carried out under magnetic stirring and argon flow for 3 h at 130 °C. The mixture was dissolved in a minimum of dichloromethane, then precipitated with methanol and dried in a vacuum oven to a constant weight.The monomer ε-caprolactone (430 mmol) was introduced into a round-bottomed flask equipped with a magnetic stirrer. Then, different volumes of 2-hydroxyethyl methacrylate were added to the reaction mixture (depending on the desired DP of PCL), and a catalytic amount of Sn(Oct)HEMA-PCL-NCO was prepared by reacting PCL with HDI in bulk . PCL (10Cellulose acetate (1 g) was dissolved in anhydrous THF (50 mL), and separately, PCL-NCO (7.5 g) was dissolved in anhydrous THF (100 mL) and an appropriate amount of DBTDL. The cellulose acetate solution was slowly added to the PCL-NCO solution while stirring for 30 min. The mixture was then stirred at 80 °C for 3 h under N2 gas atmosphere. The reaction mixture was cooled to room temperature, and then excess methanol was added to the mixture. The precipitate was recovered by vacuum filtration and the unreacted PCL-NCO was removed by Soxhlet extraction with toluene for 24 h.To evaluate the grafting of PCL onto cellulose acetate, ATR-FTIR was used. The spectra were determined on a FT/IR-4700 spectrometer . The data were collected in the spectral range of 400 to 4000 Differential scanning calorimetry (DSC)analysis, which enables determination of the latent heat of molecules during a phase transition, was performed with a TA DSC Q20 device . About 10 mg of each sample was placed in sealed aluminum capsules and subjected to 2 scans from −40 to 200 °C with a rate of 10 °C/min under inert atmosphere.−1 under 50 mL min−1 nitrogen flow within the range of 25 to 600 °C. The sample weight was between 8 and 12 mg.Standard thermogravimetric analysis (TGA)was performed using a TGAQ500 device  at a heating rate of 20 °C minThe intrinsic viscosity was measured using capillary Cannon-Ubbelohde viscometer tube  . DimethyTo prepare the stock solution, 2 g of the synthesized product was dissolved in 100 mL of DMF in an Erlenmeyer flask under stirring. Then, the solutions were prepared with concentrations varying from 1 to 2 g/dL, and the volume of each solution was 25 mL.w/w, 200 mL) were mixed and reacted at 100 °C for 3 h under N2. After being cooled to room temperature, the obtained mixturewas titrated with 0.5 NNaOH solution until neutralization.The hydroxyl value of the PCL was determined by alkaline titration after acetylation of the terminal hydroxyl groups. PCL and acetic anhydride/pyridine solution . This compact video-based instrument measures contact angles between 1° and 180° with an accuracy of ±1°. The computer software provided by KSV Instruments Ltd. precisely recorded and measured the contact angles and took pictures of the measured contact angle values. Three samples for each group were tested to calculate the average.Scanning electron microscopy was carried out with HiroxTabletop SEM SH-5500P  with an acceleration voltage of 10 kvto 15 kvin high vacuum.In the present work, CA-g-PCL copolymers were prepared by using di-isocyanated intermediates for more reactivity.A graftingto approach was utilized to covalently graft the cellulose acetate with PCL to different target DPs (CA-g-PCL). The characterization of ungrafted PCL and PCL-grafted cellulose acetate showed an increase in thermal stability (290 °C versus 340 °C), and the addition of cellulose acetate with different DPs of PCL did not affect the melting point of PCL(about 64 °C).The contact angle decreased from 86.9° for PCL to 58.2° for CA-g-PCL50 by the addition of cellulose acetate because it is hydrophilic, but by increasing the chain length of PCL, the contact angle also increased because PCL is hydrophobic.E. coli, S.aureus, and P.aeruginosa) compared withCA-g-PCL100 and CA-g-PCL200, implying that when the chain length of PCL increases, the antibacterial activity decreases because the copolymer becomes more hydrophilic.Therefore, the CA-g-PCL50 copolymer showed higher antibacterial activity against both Gram-negative and Gram-positive bacteria (


Paenibacillus polymyxa WLY78, but high concentrations of L-serine inhibit growth, nitrogenase activity, and nifH expression. Then, we revealed that expression of the serA whose gene product catalyzes the first reaction in the serine biosynthetic pathway is regulated by the T-box riboswitch regulatory system. The 508 bp mRNA leader region upstream of the serA coding region contains a 280 bp T-box riboswitch. The secondary structure of the T-box riboswitch with several conserved features: three stem-loop structures, a 14-bp T-box sequence, and an intrinsic transcriptional terminator, is predicted. Mutation and the transcriptional leader-lacZ fusions experiments revealed that the specifier codon of serine is AGC (complementary to the anticodon sequence of tRNAser). qRT-PCR showed that transcription of serA is induced by serine starvation, whereas deletion of the specifier codon resulted in nearly no expression of serA. Deletion of the terminator sequence or mutation of the continuous seven T following the terminator led to constitutive expression of serA. The data indicated that the T-box riboswitch, a noncoding RNA segment in the leader region, regulates expression of serA by a transcription antitermination mechanism.Serine is important for nearly all microorganisms in protein and downstream amino acids synthesis, however, the effect of serine on growth and nitrogen fixation was not completely clear in many bacteria, besides, the regulatory mode of serine remains to be fully established. In this study, we demonstrated that L-serine is essential for growth and nitrogen fixation of  Riboswitches, one type of non-coding RNAs, are genetic regulation elements commonly located in the 5′-untranslated regions (5′-UTRs) of mRNAs ,2,3. ManThe T-box riboswitches commonly modulate the expression of many genes encoding aminoacyl-tRNA synthetase, as well as genes encoding selected amino acid synthetase and transporter in Gram-positive bacteria ,16,17,18serA gene [serA in most members of the Firmicutes is unknown, although a T-box sequence is found in the serA regulatory region in Bacillus clausii, Bacillus halodurans, Clostridiium acetobutylicum, Clostridiium tetani, Clostridium kluyveri, and Clostridium novyi [Bacillus spp. (including B. subtilis) and other Firmicutes., serA is also regulated by the global transcriptional regulator CodY which is a sensor of nutrient availability [serA is important to bridge the pathways between glycolysis and serine biosynthesis.The amino acid L-serine (Ser) is required for protein synthesis. Serine is also a chassis material for the biosynthesis of glycine, cysteine, and tryptophan. Glycine, in turn, is a precursor for purines and heme ,31,32,33erA gene . The regum novyi ,34. Accolability , suggestnifH expression of P. polymyxa WLY78 which is a nitrogen-fixing bacterium and has great potential usage as a biofertilizer in agriculture. Then, we searched the genome sequence of the P. polymyxa WLY78 and found that a serA and a serC that are predicted to catalyze the first and the second reactions in the serine biosynthesis pathway are distributed separately. 5′-RACE (5′-Rapid Amplification of cDNA End) showed that the leader region upstream of the serA coding region is 508 bp in length and there is a 280 bp T-box region with several conserved sequences in the leader region. Alternation analysis of the three codons of serine revealed that the specifier codon is AGC. qRT-PCR analysis of the four mutants carrying the mutations in the T-box region demonstrated that the specifier codon and termination structure are essentially required for regulation of the serA expression. Our study not only reveals the regulation mechanisms of serine biosynthesis in P. polymyxa WLY78, but also will provide assistance for understanding the relationship between amino acids and nitrogen fixation.In this study, we examined the effects of L-serine on growth, nitrogenase activity, and P. polymyxa WLY78, 0–10 mM L-serine was supplemented in medium lacking nitrogen source. As shown in P. polymyxa WLY78 did not grow in medium without L-serine. It grew well in presence of L-serine and showed the best growth in the presence of 2 mM L-serine  and a CBPB gene . A serC gene is located elsewhere far from serA on the genome of P. polymyxa WLY78, but the serB gene is not found.We searched the genome of serA deletion mutant (ΔserA) of P. polymyxa WLY78 was constructed. In the minimal medium without serine, P. polymyxa WLY78 grew well, but the ΔserA mutant nearly could not grow. The ΔserA mutant grew well with the addition of serine in medium and grew best when serine concentration was 1 mM. The data suggested that serA is essential for bacterial growth in the absence of serine  method. The TSS of the serA gene is located at −508 bp upstream of the translational start site (ATG). A putative promoter (−35 region and −10 region) was identified in front of the TSS  of the  the TSS A. The daserA gene was co-transcribed with the serA coding region. The primers P1 and P2 were used to amplify a 252 bp of a double cDNA fragment (from +13 bp to +264 bp relative to TSS) that includes T-box RNA. The primers P1 and P3 were designed to amplify a 686 bp of double cDNA fragment from +13 bp to the 190th bp (relative to the translation start site ATG) in the coding region of serA that spans across leader sequence and coding region. The data indicated that T-box RNA in the leader RNA of the serA gene was co-transcribed with the serA coding region (Reverse Transcription-PCR (RT-PCR) experiments using two pairs of primers (P1 and P2) and (P1 and P3) were performed to determine that the leader region of the g region B.Ser or uncharged tRNA. Here, the secondary structure of Ser T-box of P. polymyxa WLY78 was predicted by using RNA Folding Form software online  [https://tbdb.io, accessed on 25 September 2020) [serA gene, suggesting a common regulatory mechanism involving transcription attenuation. When intracellular serine concentration is too low, UGGU within the T-box sequence may pair with the acceptor arm of uncharged tRNASer to form an antiterminator structure to derepress downstream serA expression.Generally, the T-box RNA should consist of a segment of leader RNA with conserved features that allow recognition of and pairing with the specific charged tRNAer 2020)  and the er 2020) . Ser T-blacZ coding region, and the four vectors were transformed into P. polymyxa WLY78, yielding PserA-lacZ, C87A-lacZ, T90A-lacZ, and T97A-lacZ mutants, respectively. The β-galactosidase activities of these mutants were determined when they were grown in medium containing 2 mM L-serine or without L-serine. As shown in serA-lacZ, T90A-lacZ, and T97A-lacZ mutants had much higher β-galactosidase activity in medium without L-serine than in medium containing 2 mM L-serine, indicating that mutation of UCC (from +90 bp to +92 bp) or AGU (from +95 bp to +97 bp) did not produce any impacts on regulation exhibited by serine. However, strain C87A-lacZ had only the basal β-galactosidase activity in the absence or in the presence of L-serine, suggesting that the mutation of AGC (from +85 bp to +87 bp relative to transcription start site) resulted in the loss of regulation by serine. These results indicated that Ser T-box was a bona fide serine T-box, and the specifier codon of Ser T-box was AGC residues (from +85 to +87 bp relative to transcription start site) that were complementary to the anticodon sequence of tRNASer. The data suggested that a single codon in the leader region acts as a specifier sequence, controlling the amino acid response.There are three predicted Ser codon sequences: AGC (from +85 to +87 bp relative to transcription start site), UCC (from +90 bp to +92 bp), and AGU (from +95 bp to +97 bp) in the upstream region of the 14-nucleotide T-box sequence (5′-AGGGUGGUACCACG-3′). The three Ser codon sequences AGC, UCC, and AGU were mutated to AGA (glutamate), ACC (histidine), and AGA (glutamate), respectively. A total of 606 bp, including a 188 bp promoter region preceding the TSS and a 508 bp of the leader sequence (containing the wild-type Ser codon sequence or the mutated codon sequence), was fused to the serA gene, including Stem I, Stem II, Stem III, a 14-nucleotide T-box sequence, and terminator/antiterminator helixes. In order to investigate if these motifs are involved in the regulation of serA expression, four T-box mutants with the deletion of different lengths of the T-box segments on chromosome were constructed as described in the materials and methods section. As shown in Δ216 was constructed by deleting a 216 bp fragment (from +13 to +228 relative to TSS), including the specific codon AGC for Ser (complementary to the anticodon sequence of tRNAser) and a 14-nucleotide T-box sequence. The Ser T-boxΔ70 mutant had a deletion of a 70 bp fragment (from +85 to +154 relative to TSS) including the specific codon AGC, but it still contained a 14-nucleotide T-box sequence. The Ser T-boxΔ70-BamHI mutant was constructed by changing the sequence TTTTTTT following the terminator to TGGATCC in the Ser T-boxΔ70 mutant, which was performed by using a restriction site of BamHI (GGATCC) to replace TTTTTT. The Ser T-boxΔ280 mutant deleted the 280 bp full-length of the Ser T-box riboswitch (from +1 to +280 bp relative to TSS), including TSS, a 14-nucleotide T-box sequence, and the putative terminator sequence.As described above, there are several conserved motifs in Ser T-box RNA of the serA gene of P. polymyxa WLY78 was much higher in minimal medium without serine than in medium containing 2 or 5 mM serine . The data suggest that the absence of the specific codon AGC resulted in inhibition of the serA expression. Whereas the transcripts of the serA in both Ser T-boxΔ70-BamHI and Ser T-boxΔ280 mutants were at similarly high levels whether there was serine or not. And the transcripts of serA in both mutants under the two conditions were much higher than that of P. polymyxa WLY78 in medium without serine. The data suggest that expression of serA in the two mutants was not regulated by the T-box riboswitch according to serine concentration. A common feature of the two mutants is that the terminator structure is destroyed. Thus, deletion of the terminator sequence or mutation of the continuous 7U following the terminator sequence in the two mutants led to transcription through the serA coding region.qRT-PCR analysis revealed that the transcript level of the M serine B, indicaP. polymyxa WLY78 were determined  encoding nitrogenase [P. polymyxa WLY78, the three amino acids accounted for 6.36%, 7.24%, and 0.77%, respectively, which means more glycine and cysteine demand of nitrogenase compared with the average level, and the two amino acids are synthesized from serine, though the proportion of serine of nine nitrogenase is a little lower than average level  leader sequence upstream of the serA coding region is identified. A T-box riboswitch with several conserved features: a single specifier codon for serine, three stem-loop structures, a 14 bp sequence (the T-box), and an intrinsic transcriptional terminator is found in the long leader region. There are three predicted serine codon sequences: AGC, AGU, and ACC in the leader region. Alternation of each of the three serine codons showed that only the single codon AGC (from +85 to +87 bp relative to TSS) is the specifier codon for serine, consistent with the report that there is only a single specifier sequence complementary to tRNA in the aminoacyl-tRNA synthetase genes of B. subtilis [B. clausii and B. halodurans is Ser (AGC), while for Clostridium acetobutylicum and Clostridium tetani is Ser (UCC) [Then, we investigated the regulation mechanism of the subtilis . It is per (UCC) , and theserA in the wild-type P. polymyxa WLY78 was induced by serine starvation, whereas deletion of the specifier codon (AGC) resulted in nearly no serA expression in the presence or absence of serine, suggesting that the specifier codon is essential for the serA expression. Deletion of the terminator sequence or the continuous TTTTTTT following the terminator structure led to constitutive expression of serA, suggesting that repression exhibited by the terminator is relieved. The data are consistent with the T-box riboswitch regulation system which forms the terminator or antiterminator depending on the aminoacylation state of the bound tRNA in response to amino acid starvation and to control gene expression. Also, the predicated structure of the T-box riboswitch in the leader region of the serA in P. polymyxa WLY78 has the conserved motifs as found in those of the aminoacyl-tRNA synthetase genes regulated by the T-box mechanism. Thus, our study revealed that the serA in P. polymyxa WLY78 is regulated by the T-box riboswitch control system in which a tRNA is an effector to modulate gene expression [Further, qRT-PCR showed that transcription of the pression . The preserC is located separately from serA in P. polymyxa WLY78, but there is no T-box region upstream of the serC coding region. However, sequence analysis suggests that the serC gene in Clostridiumthermocellum and Desulfitobacterium hafniense is regulated by a Ser (UCC) T-box RNA [serS encoding Seryl-tRNA synthetase is a common member of Ser T-box regulon in many Firmicutes including B. subtilis and Paenibacillus species [serB gene has not been identified in many Firmicutes including all Paenibacillus species for a long time. A recent study has revealed that ysaA encoding haloalkanoate dehalogenase may be the equivalent of serB in B. subtilis. The B. subtilis YsaA showed phosphatase activity against phosphoserine, phosphothreonine, phosphoethanolamine, and histidinol phosphate [B. subtilis ysaA to search the genome of P. polymyxa WLY78 and found that there is a ysaA gene in this bacterium.In addition, we found that a -box RNA . The var species . A serB hosphate . We haveP. polymyxa WLY78 by the Ser T-box riboswitch  at 30 °C. For nitrogenase activity assays, P. polymyxa strains were grown in a nitrogen-limited medium under anaerobic conditions. Nitrogen-limited medium used in this study contained 10.4 g/L of Na2HPO4, 3.4 g/L of KH2PO4, 26 mg/L of CaCl2·2H2O, 30 mg/L of MgSO4, 0.3 mg/L of MnSO4, 36 mg/L of ferric citrate, 7.6 mg Na2MoO4·2H2O, 10 mg/L of p-aminobenzoic acid, 5 mg/L of biotin, and 2% (w/v) glucose with 2 mM glutamate as the nitrogen source. To measure the growth, strains were grown in a mineral medium that contained 0.5 g/L KH2PO4, 1.5 g/L Na2HPO4, 1 g/L NaCl, 0.2 g/L MgSO4·7H2O, 5 g/L glucose, and 1 g/L NH4NO3, and different concentrations of L-serine was added in some experiments. When serine was the sole nitrogen source, NH4NO3 was removed. Escherichia coli strains JM109 and DH5α were used as routine cloning hosts. The thermo-sensitive vector pRN5101 was used as a gene cloning vector for gene nutation. The shuttle vector pHY300PLK that can be replicated in P. polymyxa strains and E. coli was used in the construction of lacZ fusion. When appropriate, antibiotics were added in the following concentrations: 100 μg/mL ampicillin, 5 μg/mL erythromycin, and 12.5 μg/mL tetracycline.The bacterial strains and plasmids used in this study are shown in serA) of the serA gene in P. polymyxa was constructed. Also, the three mutants  with different length deletion in the leader region of the serA gene were constructed by using a homologous recombination method, respectively. For doing this, the upstream fragment (~1 kb) and downstream fragment (~1 kb) flanking the target sequence of Ser T-box and serA were amplified by PCR using Phanta® Max Super-Fidelity DNA Polymerase , respectively. The two fragments were then fused with the HindIII/BamHI digested pRN5101 vector using Gibson assembly master mix , generating four recombinant plasmids: pRDSer T-boxΔ216, pRDSer T-boxΔ70, pRDSer T-boxΔ280, and pRDserA. The four recombinant plasmids were individually transformed into P. polymyxa WLY78 as described by Wang et al. [r). Subsequently, the double-crossover transformants were selected from the initial erythromycin transformants after several rounds of nonselective growth at 39 °C and confirmed by PCR amplification using corresponding primers and Green Taq Mix . The primers used here are listed in The in-frame-deletion mutant  in the leader region of the serA gene were replaced by a restriction site of BamHI (GGATCC) by primer design, the upstream fragment (~1 kb) and downstream fragment (~1 kb) flanking the region containing the continuous T sequences were amplified by PCR from genome DNA of Ser T-boxΔ70. The two fragments were then fused to the HindIII/BamHI digested pRN5101 vector using Gibson assembly master mix, generating the recombinant plasmid pRUBamHI. The recombinant plasmid was transformed into the Ser T-boxΔ70 mutant. The single-crossover transformants were selected for erythromycin resistance (Emr). Subsequently, the double-crossover transformants were selected from the initial Erythromycin resistance transformants after several rounds of nonselective growth at 39 °C. Then PCR amplification and subsequent digestion with BamHI were used to confirm the mutants. The primers used here are listed in The mutant Ser T-boxP. polymyxa WLY78 using primers Ser T-boxpU-f and Ser T-boxp-r to construct PSer-lacZ. The lacZ coding region was amplified from the plasmid pPR9TT with primers lacZ-f and lacZ-r. The site mutation of specific codon was based on PSerA-lacZ using primers of MutC87A-f/MutC87A-r, MutT90A-f/MutT90A-r, and MutT97A-f/MutT90A-r by PCR, respectively. The PCR product was digested by DpnI then transformed to JM109 competent cells to select positive mutation transformants by sequencing. The successful site mutation fragment was PCR amplified and assembled to pHY300PLK and then transformed into P. polymyxa WLY78.A total of 696 bp fragments, including a 188 bp promoter region preceding the TSS (transcription start site) and a 508 bp DNA fragment preceding the of ATG (translation start site) was amplified from the genomic DNA of The leader region was confirmed by reverse transcription PCR (RT-PCR), and transcription levels of genes were confirmed by quantitative real-time PCR (qRT-PCR). For corresponding experiments, 50 mL of cultures were harvested at 4 °C and rapidly frozen under liquid nitrogen. Total RNAs were extracted with RNAiso Plus  according to the manufacturer’s protocol. Synthesis of cDNA was performed using PrimeScript RT reagent Kit with gDNA Eraser . For RT-PCR 0.5 μg cDNA was used. qRT-PCR was performed on Applied Biosystems 7500 Real-Time System with the following program: 95 °C for 15 min, 1 cycle; 95 °C for 10 s, and 65 °C for 30 s, 40 cycles. The relative expression level was calculated using the ΔΔCt method, each experiment was performed in triplicate. Primers used for qRT-PCR are listed in P. polymyxa WLY78 and its mutant strains were grown in 5 mL of LD medium overnight. The cultures were collected by centrifugation, washed three times with sterilized water, and then resuspended in a nitrogen-limited medium containing 2 mM glutamate as a nitrogen source to a final OD600 of 0.2–0.4. Then, 4 mL of the culture was transferred to a 25-mL test tube and the test tube was sealed with a rubber stopper, L-serine was added to proper concentrations at the same time for special experiments. The headspace in the tube was then evacuated and replaced with argon gas. Then, approximately 2 mL of C2H2 (10% of the headspace volume) was injected into the test tubes. After incubating the cultures at 30 °C for the corresponding time, a 100 μL gas sample was taken out and injected into gas chromatography to quantify ethylene (C2H4) production. The nitrogenase activity was expressed in nmol C2H4/mg protein/hr. All treatments were in three replicates and all the experiments were repeated three or more times.Acetylene reduction assays were performed as described previously to measure nitrogenase activity . P. poly®-T vector  after adding A tail by taq DNA polymerase  and then sequenced.The 5′-RACE method was used to determine the transcription start site (TSS) using the SMARTer RACE cDNA Amplification Kit . Gene-specific primers are listed in


Atrial fibrillation (AF) is the most common cardiac arrhythmia in patients with chronic kidney disease (CKD) and acute coronary syndrome (ACS). This study aimed to explore the frequency and impact of AF on clinical outcomes in CKD patients with ACS.CKD inpatients with ACS between November 2014 and December 2018 were included based on the improving care for cardiovascular disease in China-ACS (CCC-ACS) project. Included patients were divided into an AF group and a non-AF group according to the discharge diagnosis. Multivariable logistic regression was used to adjust for potential confounders.2, and 764 had an estimated glomerular filtration rate (eGFR) < 45 ml/min/1.73 m2. Compared with the non-AF group, the AF group had a higher risk of in-hospital mortality  and major adverse cardiovascular events (MACEs) . We also found that compared with patients with eGFR 45 to < 60 ml/min/1.73 m2, patients with eGFR < 45 ml/min/1.73 m2 had a 1.512-fold increased risk of mortality and a 1.435-fold increased risk of MACEs.A total of 16,533 CKD patients with ACS were included. A total of 1418 (8.6%) patients had clinically recognized AF during hospitalization, 654 of whom had an eGFR of 45 to < 60 ml/min/1.73 mAF was a risk factor affecting the short-term prognosis of ACS patients in the CKD population. Furthermore, the lower the eGFR, the higher the risk of in-hospital mortality and MACEs in CKD patients with ACS.Trial registry: Clinicaltrial.gov, NCT02306616. Registered 29 November 2014, https://clinicaltrials.gov/ct2/show/NCT02306616?term=NCT02306616&draw=2&rank=1The online version contains supplementary material available at 10.1186/s12872-021-02125-z. Chronic kidney disease (CKD) has become a global public health problem , 2. In 2This article was a retrospective study based on the Improving Care for Cardiovascular Disease in China-Acute Coronary Syndrome (CCC-ACS) project, which is a national, hospital-based quality improvement project with an ongoing database, aiming to increase adherence to ACS guidelines in China and to improve patient outcomes. The project was launched in 2014 as a collaborative initiative of the American Heart Association (AHA) and the Chinese Society of Cardiology (CSC). A total of 92,509 patients from 246 hospitals were recruited, representing the diversity of ACS care in hospitals in China. Clinical data were collected via a web-based data collection platform . Data elements collected in this study included patient demographics, medical history, symptoms on arrival, in-hospital treatments, discharge medications, and secondary prevention strategies. The eligible patients each month were consecutively entered into the online data reporting system before the middle of the following month after patient discharge. The following four approaches were adopted to ensure the accuracy and completeness of the data: (1) face-to-face training workshops, (2) use of a standardized online reporting tool, (3) onsite quality control, and (4) monitoring of data completeness. Details of the design and methodology of the CCC-ACS project have been published previously .2. CKD severity was then categorized according to the KDIGO guidelines as follows . However, prior AF was significantly related to in-hospital MACEs . We also found that compared with the patients with eGFR 45 to < 60 ml/min/1.73 m2, patients with eGFR < 45 ml/min/1.73 m2 had a 1.512-fold increased risk of mortality  –11. In tIn our study, AF was defined according to the discharge diagnosis, which made it more accurate in terms of the patients’ characteristics. The results showed that the risk of in-hospital mortality and MACEs was significantly higher in the AF group than in the non-AF group. Moreover, after adjusting for confounders, AF was found to be an independent risk factor for in-hospital mortality and MACEs in CKD patients with ACS. Further analysis showed that new-onset AF was significantly related to in-hospital mortality and MACEs. However, prior AF was significantly related to in-hospital MACEs, not related to mortality. Furthermore, we analyzed the relationship between renal function and in-hospital prognosis in CKD patients with ACS. The results showed that the decrease in renal function was an independent risk factor for adverse in-hospital outcomes in CKD patients with ACS. These patients, especially those with severe renal insufficiency, should be given more attention and closer monitoring in clinical practice to reduce the incidence of in-hospital mortality and MACEs.2, which is different from the KDIGO criteria. Finally, due to the observational nature of the study, causality and the underlying mechanisms could not be determined.Our study also had some limitations. First, since this study included observational data from a large database, although we adjusted for a wide range of potential confounders, we cannot rule out residual confounding factors, including whether the AF was new-onset or pre-existing and the type, occurrence time and duration of AF. Second, because of the atypical symptoms of some patients with AF, there may be some cases of undiagnosed AF. Third, due to the limitation of data collection, the definition of CKD in our study only included eGFR < 60 ml/min/1.73 mIn summary, our study was the first and largest to investigate ACS with concomitant AF in CKD patients in China. In this study, we found that AF was a risk factor affecting the short-term prognosis of ACS patients in the CKD population. Furthermore, the lower the eGFR, the higher the risk of in-hospital mortality and MACEs in CKD patients with ACS. These patients, especially those with severe renal insufficiency, should be given more attention and closer monitoring in clinical practice. Further studies are needed to investigate the effect of AF on the long-term prognosis of CKD patients with ACS.Additional file 1. Table S1. Treatments pre-hospital and during hospitalization. Table S2. Investigators of CCC-ACS project.


SARS-CoV-2 infections elicit a humoral immune response capable of neutralising the virus. However, multiple variants have emerged with mutations in the spike protein amongst others, the key target of neutralising antibodies. We evaluated the neutralising efficacy of 89 serum samples from patients, infected with SARS-CoV-2 in the beginning of 2020, against two virus variants isolated from acutely infected patients and harbouring spike protein mutations. One isolate was assigned to lineage B.1.351 (MUC-IMB-B.1.351) whilst the other (MUC-484) was isolated from an immunocompromised patient, sharing some but not all mutations with B.1.351 and representing a transitional variant. Both variants showed a significant reduction in neutralisation sensitivity compared to wild-type SARS-CoV-2 with MUC-IMB-B.1.351 being almost completely resistant to neutralisation. The observed reduction in neutralising activity of wild-type-specific antibodies against both variants suggests that individual mutations in the spike protein are sufficient to confer a potent escape from the humoral immune response. In addition, the effect of escape mutations seems to accumulate, so that more heavily mutated variants show a greater loss of sensitivity to neutralisation up to complete insensitivity as observed for MUC-IMB-B.1.351. From a clinical point of view, this might affect the efficacy of  antibody treatment of patients with prolonged infections as well as patients infected with variants other than the donor. At the same, this could also negatively influence the efficacy of current vaccines (as they are based on wild-type spike protein) emphasising the need to thoroughly surveil the emergence and distribution of variants and adapt vaccines and therapeutics accordingly. SARS-CoV-2 has steadily changed its genome through mutations during its global spread amongst humans. Such mutations can in principle affect the entire viral genome. Of particular interest, though, are those that occur in the spike (S) protein are of particular interest, since this protein is responsible for binding to the human ACE2 receptor and is thus indispensable for virus cell entry. It is therefore not surprising that the S protein and in particular its receptor binding domain has been identified as the key target for eliciting potent neutralising antibodies (NAbs), likely protecting against reinfection , 2. MoreHowever, several novel variants that have recently emerged harbour a number of mutations, some of which are in the gene encoding the S protein . This haLineage B.1.351 (Beta), also known as 20H/501Y.V2, is one of multiple SARS-CoV-2 variants circulating globally and is classified as a VOC by the WHO . It was MUC-484 has several mutations, four of which are located within the S protein, including the D614G mutation. Most notably, the strain also acquired the E484K mutation present in B.1.351. However, it contains neither the above-mentioned N501Y nor the K417N mutation, making it an interesting variant to investigate the influence of E484K in absence of N501Y and K417N compared with the more recent line B.1.351, which contains all three mutations.As previously described , all samReplication competent SARS-CoV-2 was isolated from a nasopharyngeal swab of a patient diagnosed with COVID-19 by quantitative PCR with reverse transcription (RT-qPCR). Whole genome sequencing of this isolate (from now on referred to as MUC-IMB-B.1.351) was performed in accordance with the German Regulation for molecular genetic surveillance of the coronavirus SARS-CoV-2. The second isolate (from now on referred to as MUC-484) derived from an immunocompromised patient and was previously described . LollipoBoth viruses were grown on Vero E6 cells. Viral stocks were prepared, titrated and stored at − 80 ℃ until further use.4 cells/50 µl) were added to each well. After 72 h (37 ℃), supernatants were discarded and wells were fixed  and stained with 0.1% crystal violet. The NAb titre corresponded to the reciprocal of the highest sample dilution showing complete inhibition of CPE. A virus re-titration was performed in triplicates on every plate and exact titres were determined by retrograde calculation.SARS-CoV-2 NAb titres were determined as previously described . In brieWe were able to generate a high-quality genome from a patient sample containing replication competent SARS-CoV-2. The isolated genome holds 29,903 nucleotides in length, which equals 100% of the SARS-CoV-2 genome. The theoretically obtained sequencing depth was 1536-fold. Sequencing analysis revealed 26 nonsynonymous mutations relative to the Wuhan sequence, assigning the isolate to the lineage B.1.351. An overview of all mutations is shown in Fig. As shown in Fig. p < 0.0001 by Wilcoxon). At the same time, all samples (16/16) that initially tested negative for NAbs against WT virus were also unable to neutralise MUC-IMB-B.1.351  was unable to neutralise MUC-IMB-B.1.351. For the nine samples that partly retained their neutralising ability, a notable reduction was observed with a median decrease of 91.5% in neutralising activity. Altogether, the mean loss of neutralising activity against MUC-IMB-B.1.351 was 99% and the observed decrease in titres was statistically significant  between the NAb titre against WT SARS-CoV-2 and the titre against MUC-IMB.1.351 of the same sample. .Direct comparison of NAbs titres against WT and MUC-484 showed that the majority of the tested serum samples (∑ = 80) was unable to retain their neutralising efficacy. Of the 64 samples that were able to neutralise MUC-IMB-1 only one-third (20/64) was also able to neutralise MUC-484. At the same time, a notable reduction in NAbs titres was observed for 18 of these samples with a median decrease of 89.6%. Two samples showed a slight increase in neutralisation when tested against MUC-484. A total of 44 samples showed no neutralisation when tested against MUC-484 although they were able to neutralise WT SARS-CoV-2 in parallel control tests. Altogether, the median loss of neutralising efficacy was 90.6%. All 16 samples that initially tested negative for NAbs against WT virus were also unable to neutralise MUC-484  between the NAb titre against WT SARS-CoV-2 and the titre against MUC-IM-B.1.351 of the same sample . Interestingly, we observed the same phenomenon with one serum when tested against B.1.1.7 .The fact that the E484K emerged independently in an immunocompromised patient with prolonged infection under plasma donation treatment  and is hThe fact that no prediction could be made about the neutralising efficacy of a serum against either variant based on the NAbs titre of the same sample against WT SARS-CoV-2 must be considered, especially for the therapeutic use of convalescent plasma. Ideally, the neutralising efficacy of convalescent plasma should be determined against the major virus clades circulating at that time and administered to patients infected with a well-neutralised clade whenever possible. This includes that NAbs detection assays such as virus neutralization tests (VNT) or surrogate tests should be continuously adapted to cover current virus clades.A potential limitation of our study is its relatively low number of tested samples as well the overall low titres of these samples, which might lead to an overestimation of the variants immune escape potential In addition, we could not include in our investigations the potentially pre-existing cellular immune response, which might be more cross-reactive than NAbs. Thus, future studies focussing on the cellular immune response are needed.Overall, our study highlights the humoral immune evasion potential not only of globally circulating VOCs such as B.1.351 but also of variants emerging in patients under plasma therapy. Mutations of the S protein are of particular interest and we demonstrate that different mutations and combinations thereof can be associated with a reduction up to complete loss of neutralising antibody cross-reactivity against novel emerging viral strains.


Cotyledon orbiculata, commonly known as pig’s ear, is an important medicinal plant of South Africa. It is used in traditional medicine to treat many ailments, including skin eruptions, abscesses, inflammation, boils and acne. Many plants have been used to synthesize metallic nanoparticles, particularly silver nanoparticles (AgNPs). However, the synthesis of AgNPs from C. orbiculata has never been reported before. The aim of this study was to synthesize AgNPs using C. orbiculata and evaluate their antimicrobial and immunomodulatory properties. AgNPs were synthesized and characterized using Ultraviolet-Visible Spectroscopy (UV-Vis), Dynamic Light Scattering (DLS) and High-Resolution Transmission Electron Microscopy (HR-TEM). The antimicrobial activities of the nanoparticles against skin pathogens  as well as their effects on cytokine production in macrophages (differentiated from THP-1 cells) were evaluated. The AgNPs from C. orbiculata exhibited antimicrobial activity, with the highest activity observed against P. aeruginosa (5 µg/mL). The AgNPs also showed anti-inflammatory activity by inhibiting the secretion of pro-inflammatory cytokines  in lipopolysaccharide-treated macrophages. This concludes that the AgNPs produced from C. orbiculata possess antimicrobial and anti-inflammation properties. Cotyledon orbiculata is a succulent plant indigenous to Southern Africa and is commonly found in the Western Cape province. It is a small shrub, commonly known as pig’s ear, that has thick green fleshy leaves with a red line around the edges of the leaves [C. orbiculata have many biological activities, which include antibacterial, antifungal, anticonvulsant, antinociceptive, anti-inflammatory, antihelmintic and antioxidant activities [C. orbiculata plant against Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Bacillus subtilis and Candida albicans. All the extracts showed antimicrobial activity, with Minimum Inhibitory Concentration (MIC) values ranging from 1.6 to 12.5 mg/mL [C. orbiculata leaf significantly attenuated carrageenan-induced oedema in the paws of rats, and thus demonstrated the anti-inflammatory properties of the plant extract [e leaves . It is ue leaves ,3. The fe leaves . Studiestivities ,5,6,7. A extract .Galenia African; Hypoxis hemerocallidea [Terminalia mantaly [Capparis spinosa [Datura stramonium [Ocimum tenuiflorum; Citrus sinensis; Solanum tricobatum; Centella asiatica; Syzygium cumini [Musa paradisiacal [Aspalathus hispida; Aspalathus linearis; Asparagus rubicundus; Cynanchum africanum; Dicerothamnus rhinocertis; Eriocephalus africanus; Hermannia alnifolia; Indigofera brachystachya; Lobostemon glaber; Metalasia muricate; Nidorella foetida; Otholobium bracteolatum; Podocarpus falcatus; Podocarpus latifolius; Salvia africana-lutea; Senecio pubigerus; Searsia dissecta; Camellia sinensis; and Camellia sinensis [Green nanotechnology is a division of nanotechnology which involves the synthesis of biogenic nanomaterials using eco-friendly methods that are safe and cost effective . Microorcallidea ; Termina mantaly ; Cappari spinosa ; Datura ramonium ; Ocimum m cumini ; Musa padisiacal ; Aspalatsinensis .Cyclopia intermedia plant, can be used to produce gold nanoparticles, which have similar physicochemical properties to those produced using the water extract of C. intermedia [C. orbiculata extracts contain flavonoids, phenols, saponins, tannins, triterpene steroids and reducing sugars, which contribute to its biological activities [C. orbiculata extracts may have antibacterial and immunomodulatory effects.It is likely that the phytochemicals that are responsible for the bioactivities of medicinal plant extracts could also be involved in the synthesis of the biogenic metal nanoparticles. A study by Aboyewa et al. 2021) demonstrated that the compound mangiferin, which is a xanthonoid that is found abundantly in extracts of the termedia . The nan demonstrNanomaterials are widely used in different biomedical applications and research. They are incorporated in targeted therapy, drug delivery and in the diagnosis of various diseases . Among mH. hemerocallidea plant have been shown to possess anti-inflammatory properties by reducing the levels of several pro-inflammatory cytokines  [Inflammation is an essential response triggered by the body’s immune systems to fight infections. However, chronic and uncontrolled inflammation in diseases such as cancer, rheumatoid arthritis, asthma and diabetes is associated with adverse effects. Pro-inflammatory cytokines promote inflammation; however, excessive or uncontrolled production of these cytokines can lead to the development of chronic inflammatory conditions such as chronic obstructive pulmonary disorder, osteoarthritis and cancer ,22. Biogd IL-1β) .C. orbiculata aqueous extract to synthesize AgNPs. The reaction parameters of AgNPs synthesis such as time, temperature, silver nitrate (AgNO3) and plant extract concentrations were studied in order to optimise the synthesis of the AgNPs from C. orbiculata (Cotyledon-AgNPs). The physicochemical properties of the Cotyledon-AgNPs were characterised. The antimicrobial activity against common skin pathogens , Pseudomonas aeruginosa and C. albicans) and the immunomodulatory effects of Cotyledon-AgNPs were determined. Results obtained from this study could potentially lay a foundation for the discovery of new antimicrobial and anti-inflammatory drugs. This study investigated the use of C. orbiculata plants were purchased from Van Der Berg Garden Village nursery in Stellenbosch, Cape Town, South Africa. AgNO3 (99%) was obtained from ACE chemicals . Alamar blue was obtained from ThermoFisher Scientific . Lipopolysaccharides (LPS), phorbol 12-myristate 13-acetate (PMA), WST-1 reagent, Roswell Park Memorial Institute (RPMI) medium, Müller-Hinton broth (MHB), Yeast Peptone broth (YPB), Luria-Bertani broth (LB broth), Phosphate-buffered saline (PBS), Fetal bovine serum (FBS), Pen-strep (penicillin and streptomycin), 0.45 µm syringe filters and Whatman filter paper No.1 were obtained from Sigma-Aldrich . The cytokine ELISA kits were from Bioo Scientific, PerkinElmer .Fresh C. orbiculata leaves were cut from the whole plant and washed with distilled water. The fresh leaves (300 g) were then cut into small pieces, blended and macerated in 600 mL of distilled water overnight. Thereafter, the extracts were filtered using Whatman filter paper No.1, followed by microfiltration using 0.45 µm filters. The extract was then concentrated by freeze drying and stored at 4 °C until use.The fresh Cotyledon-AgNPs was performed according to the methodology reported before, with modifications [3 solution (1 mM and 3 mM) was mixed with 1 mL of the different concentrations of plant extract in glass tubes . These mixtures were incubated at 25 °C (room temperature) or 70 °C while stirring for 0.5, 1, 2 or 3 h in the dark. The Cotyledon-AgNPs were then purified by centrifugation at 10,000 rpm , Eppendorf AG, Hamburg, Germany) for 10 min, and then the pellet was re-suspended in distilled water. This process was repeated three times.The synthesis of ications . In shorCotyledon-AgNPs was evaluated in different biological media and buffers used in this study . This assay was performed immediately after the Cotyledon-AgNPs were purified. In glass test tubes, 250 µL of aqueous solutions of Cotyledon-AgNPs were mixed with the same volume of the medium or buffer. The tubes were incubated at 25 °C or 37 °C for 24 h. The stability of these nanoparticles was evaluated by measuring changes in their Ultraviolet-Visible spectroscopy (UV-Vis) spectra.The stability of the Cotyledon-AgNPs was confirmed by observing their Surface Plasmon Resonance (SPR) peaks using UV-Visible spectroscopy ) at a wavelength range of 250 to 800 nm.The formation of Cotyledon-AgNPs.The DLS analysis was performed using Zetasizer Nano ZS90  to determine particle size distribution, zeta potential and Polydispersity Index (PDI) values of the Cotyledon-AgNPs were determined by HR-TEM analysis using a FEI Tecnai G2 F20 field-emission gun . For the preparation for HR-TEM, a drop of Cotyledon-AgNPs solution was loaded onto a carbon coated copper grid. The grid was dried under a Xenon lamp for 10 min before analysis. The particle size distribution was determined using Image J software. Selected Area Electron Diffraction (SAED) and Energy-dispersive X-ray spectroscopy (EDX) analyses were also analysed on the same samples using HR-TEM.The morphology and the particle size distribution of Cotyledon-AgNPs were evaluated by determining their MIC using a microdilution assay [S. aureus (ATCC 25923), MRSA (33591), S. epidermidis (ATCC 12228), P. aeruginosa (ATCC 27853) and C. albicans (ATCC 10231). Briefly, 50 μL of Cotyledon-AgNPs (in LB broth) were added in 96-well plates with decreasing concentrations .The antimicrobial properties of the on assay . The MIC8 CFU/mL) [Microbial suspensions of the bacteria and the fungus were cultured in LB broth and diluted until they reached a 0.5 McFarland standard (standard is approximately 1 × 10 CFU/mL) . A volumAfter incubation, 10 µL of Alamar Blue was added to each well and the plate was incubated for 3 h in the dark. In the presence of viable bacteria, the non-fluorescent Alamar Blue dye (resazurin) was reduced to resofurin, a pink compound that was highly fluorescent . TherefoThis assay was conducted according to the previously reported methodology, with modifications . The MBC2 in a SL SHEL LAB incubator .The human monocytic leukaemia cell line, THP-1, was obtained from ATCC (TIB-202), . THP-1 cells were cultured in RPMI (1640) medium containing 10 % FBS and 1 % Pen-strep. The cells were incubated at 37 °C in a humidified atmosphere of 5 % CO5 cells/mL and were treated with 100 nM PMA. These cells were incubated at 37 °C in a humidified atmosphere of 5 % CO2. After 3 days of incubation, the culture media was replaced with PMA-free media (resting phase) for another 24 h [THP-1 monocytes were differentiated into macrophages using PMA. The THP-1 monocytes were seeded in 24-well plates (500 µL) at a density of 2 × 10her 24 h .Cotyledon-AgNPs on the differentiated THP-1 macrophages. Using 96-well cell culture plates, the macrophages were treated with different concentrations of the Cotyledon-AgNPs . The cells were incubated for 24 h at 37 °C in a humidified atmosphere of 5 % CO2. After incubation, the medium was removed from the wells and replaced with a medium containing 10 % WST-1 reagent. The cells were incubated for another 3 h, and the absorbance of the culture media was measured at 440 nm (reference 630 nm) using the POLARstar Omega plate reader . Cell viability was expressed as a percentage of the absorbance of treated cells to control (untreated) cells.Cell viability was evaluated using the WST-1 assay, according to the manufacturer’s protocol. This assay was conducted to determine the toxicity of the Escherichia coli 0111:B4. PMA-differentiated THP-1 macrophages were stimulated with 1 µg/mL LPS [Cotyledon-AgNPs at 37 °C for 24 h. The LPS control was treated with LPS only. After 24 h incubation, the cell supernatants were transferred into Eppendorf tubes and centrifuged at 1500 rpm for 10 min. The supernatants were then collected and stored at −80 °C until measurement of cytokines. The production levels of the cytokines, IL-1β, IL-6 and TNF-α were determined using enzyme-linked immunosorbent assay (ELISA) kits, according to the manufacturers’ instructions.Stimulation of macrophages for cytokine determination was performed using LPS from g/mL LPS  for 6 h Cotyledon-AgNPs were determined using the two-way analysis of variance (ANOVA). Values were considered to be statistically significant at p < 0.0001.Statistical analysis of the data was conducted using GraphPad Prism 6 software. The results were expressed as mean ± standard error of the mean (SEM). The significance of the cell viability and immunomodulatory effects of + to Ag0) [Cotyledon-AgNPs was optimised by varying different reaction parameters such as temperature, time and the concentration of both AgNO3 and the plant extract solutions.Generally, AgNPs are formed by the reduction of silver salt to silver ions (Ag to Ag0) . The phy to Ag0) . Plants 3 and C. orbiculata aqueous extract solution). Immediately after the addition of the plant extract to the AgNO3 solution, the mixture was colourless (The synthesis of AgNPs was indicated by a colour change in the reaction mixture (AgNOlourless A. Howevemax) of the Cotyledon-AgNPs was around 420 nm, which is characteristic of AgNPs. AgNPs exhibit an SPR at around 400–500 nm [The synthesis of AgNPs was further confirmed by measuring the UV-Vis spectra between 250–800 nm. The maximum absorbance (λ0–500 nm . SPR of 0–500 nm .Cotyledon-AgNPs was optimized using two AgNO3 concentrations (1 mM and 3 mM). These two concentrations are commonly reported in the literature for the synthesis of AgNPs from plants [3 concentration on the synthesis of Cotyledon-AgNPs. The use of 3 mM AgNO3 produced AgNPs with an intense brown colour and sharper absorbance peaks. Moreover, 3 mM AgNO3 produced a larger amount of AgNPs. This was evident from the height of the absorbance peaks, which gives an indication of the number of the nanoparticles produced. It is known that absorbance is directly proportional to the concentration of AgNPs in a solution [3.The synthesis of m plants ,34,35,36solution . Subsequ3 (3 mM). This further suggests that extremely high concentrations of the plant extract hinder the formation of nanoparticles.Another reaction parameter evaluated was the concentration of the plant extract. Of the different plant extract concentrations tested  only three concentrations  gave nanoparticles, as determined by the change in the colour of the respective reaction mixtures after the addition of AgNOmax suggest the formation of smaller and monodispersed nanoparticles by increasing the concentrations of the plant extract [C. orbiculata extract concentration (from 1.5 to 6 mg/mL) enhanced the formation of AgNPs.It can be observed in  extract . A study extract . This isC. orbiculata extract produced AgNPs with an absorbance reading of 1.47 after 1 h from the start of the reaction, whereas 1.5 mg/mL of the extract produced AgNPs with a lower absorbance reading of 0.66 within the same time frame. This might be because of the availability of higher amounts of phytochemicals when using high concentrations of the plant extract, which, therefore, increased the reduction power in the reaction medium, leading to a reduction of more AgNO3 molecules in a short time [Increasing the plant extract concentrations also changed the intensity of the reaction mixture’s colour, and produced AgNPs at a faster rate than the lower concentrations of the plant extract. This is as also indicated by the increase in the absorbance of the UV-Vis spectra. As shown in ort time ,38,39. TC. sinensis (sweet orange) peel extract showed a decrease in the size of AgNPs with increasing temperature [C. sinensis aqueous peel extract produced particles with a size of 35 nm but, at 60 °C, particle size decreased to 10 nm. In this study, however, no nanoparticles were produced at 25 °C. The synthesis was only accomplished at 70 °C. This is also in agreement with a study by Elbagory and colleagues (2016), in which aqueous extracts of Asparagus rubicundus, Aspalathus hispida and Dicerothamnus rhinocertis produced gold nanoparticles at 70 °C only [Temperature is another important factor in the synthesis of nanoparticles. High temperature is suggested to facilitate the synthesis of nanostructures . It has perature . At 25 °eagues 20, in whic °C only .Cotyledon-AgNPs synthesis , but also at 37 °C to observe the effect of biological assay conditions on their stability. Incubation of most biological assays, such as cell culture, cell viability assays and bacterial culture, is conducted under these temperatures. The UV-Vis absorption spectra showed that the SPR characteristics of the nanoparticles did not change throughout the incubation time in RPMI, LB broth and PBS, implying that they are stable in these solutions. On the other hand, the Cotyledon-AgNPs were not stable in MHB and YPB at both temperatures . The antimicrobial activity of Cotyledon-AgNPs compared extremely well with the positive controls (ampicillin and fluconazole). In fact, the antimicrobial activity of Cotyledon-AgNPs against MRSA and P. aeruginosa was higher than both controls used. While the MIC for ampicillin was 310 µg/mL for MRSA and >1000 µg/mL for P. aeruginosa, the MICs for Cotyledon-AgNPs were 40 and 5 µg/mL against MRSA and P. aeruginosa, respectively.The antimicrobial activity of Cotyledon-AgNPs were more active against the Gram-negative bacteria (P. aeruginosa) as compared to the Gram-positive bacteria . This is likely due to the different membrane structures and compositions in the cell wall of the microorganisms [Cotyledon-AgNPs also exhibited better antibacterial activity than the antifungal activity, which corresponds to the findings reported previously [The rganisms . Gram-porganisms . These rrganisms ,13,15. Ieviously .The high antibacterial activity of AgNPs is attributable to their large surface area, which provides better contact of the nanoparticles with the cell wall of microorganisms . Other mThe human monocytes THP-1 cells, used herein to evaluate immunomodulatory activity, were cultured in RPMI and differentiated into macrophages using 100 nM of PMA. PMA, a cognate of diacylglycerol, is an activator of protein kinase C that is used in cell differentiation ,76. UponCotyledon-AgNPs on the differentiated THP-1 macrophages. This assay is based on the conversion of a water-soluble tetrazolium salt into formazan by the mitochondrial dehydrogenases of the viable cells. Therefore, the number of viable cells can be quantified by detecting the amount of formazan produced [Cell viability assays are crucial for the determination of the cytotoxic effects of compounds on different cell lines in vitro. In this study, a WST-1 assay was used to determine the cytotoxicity of produced .Cotyledon-AgNPs showed no toxicity to THP-1 cells at concentrations below 10 µg/mL  to the macrophages reduced the production levels of these cytokines. The levels of cytokine production in the cells treated with Cotyledon-AgNPs was significantly decreased by approximately 3.5-, 7- and 10.5-fold for TNF-α, IL-1β and IL-6, respectively, when compared to the LPS treated cells  in THP-1 cells, suggesting that they have some anti-inflammatory activity [ELISA was used to determine the effects of bacteria . Exposurbacteria . The leved cells . Therefoactivity ,83. C. orbiculata aqueous extract. The synthesis was optimized by varying reaction parameters such as time, temperature, AgNO3 and plant extract concentrations. The formulated Cotyledon-AgNPs were fully characterized and were found to be stable in different biological media. The antimicrobial study showed that Cotyledon-AgNPs exhibited antimicrobial activity against several skin-related pathogens more than C. orbiculata aqueous extract. Cotyledon-AgNPs also exhibited anti-inflammatory effects by suppressing the production of pro-inflammatory cytokines  in macrophages.AgNPs were successfully synthesized using Cotyledon-AgNPs as antimicrobial agents in the treatment of skin infections. The study also shows the potential of Cotyledon-AgNPs as agents to control inflammatory disorders that cause chronic inflammation.Therefore, this study demonstrates the potential of